Transcript Chapter 12. Protein biosynthesis (P215, sP875)

Chapter 12. Protein biosynthesis
Protein biosynthesis is a process to express
genetic information in living cells, which is
called translation.
The genetic information flows as:
Transcription
DNA
Reverse
transcription
Translation
RNA
Protein
1. Components of Protein Biosynthesis
Protein biosynthesis requires: amino acids,
mRNA, tRNA, ribosomes, protein factors,
and synthetic enzymes.
1) Messenger RNA: a template for protein
biosynthesis, which is read in a 5’3’
direction. Each three nucleotides form a
codon representing for a specific amino
acid. Thus, the base sequence of an mRNA
molecule determines the amino acid
sequence of the protein.
Codons in mRNA
U
C
A
G
U
C
A
G
Phe
Phe
Leu
Leu
Ser
Ser
Ser
Ser
Tyr
Tyr
Stop
Stop
Cys
Cys
Stop
Trp
U
C
A
G
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
His
His
Gln
Gln
Arg
Arg
Arg
Arg
U
C
A
G
Ile
Ile
Ile
Met
Thr
Thr
Thr
Thr
Asn
Asn
Lys
Lys
Ser
Ser
Arg
Arg
U
C
A
G
Val
Val
Val
Val
Ala
Ala
Ala
Ala
Asp
Asp
Glu
Glu
Gly
Gly
Gly
Gly
U
C
A
G
mRNA in eukaryotes is usually
monocistronic: one mRNA encodes only a
single polypeptide chain.
mRNA in prokaryotes usually encodes more
than one polypeptide chain. This is called
polycistronic.
A) Degeneracy of codons: refers to the fact
that an amino acid has more than one
codon.
 one of the consequences of degeneracy is
that a mutation which produces a base
change in DNA may not result in an amino
acid change in the encoded protein.
 Synonyms: refers to the codons for the
same amino acid. e.g. GUU, GUC, GUA,
GUG represent for Val.
B) Universility of codons: this genetic code
system is used by all living organisms
except in some cases:
in cytosol
in mitochondria
AUA
UGA
AGA
CGG
CUN
Ile
Stop
Arg
Arg
Leu
Met
Trp
Stop (animal)
Trp (plant)
Thr (yeast)
C) Reading frames: refer to the different
combinations for each three nucleotides that
are read as a codon: each mRNA sequence
can be read in three possible reading
frames.
Reading frame 1: UUA UGA GCG CUA AAU
Leu Stop Ala
Leu Asn
Reading frame 2: U UAU GAG CGC UAA AU
Tyr Glu Arg Stop
Reading frame 3: UU AUG AGC GCU AAA U
Met Ser Ala Lys
D) Open reading frames: refer to the runs of
codons that start with ATG and end with
TGA, TAA, or TAG. The open reading
frames can be used to predict the protein
sequence encoded.
2) Transfer RNAs: the fidelity of protein
biosynthesis requires tRNAs to serve as
adapters that can recognize the
correspondent codons and carry amino acids
to the right positions in translation.
Each tRNA only brings with it an amino
acid, and recognizes and binds to a specific
codon.
Secondary structure of tRNA
3
A
A
C
A
C
A
5
A
A
A
A
A
A
A
A
A
A
A
TC loop
U
C
A
A
A
A
A
A
A
A
A
G
A
T
C
鴠
A
G
A
A
A
A
G
extra arm
A
DHU loop
U
A
Anticodon loop
Tertiary structure of tRNA
Codon-anticodon interaction by
base pairing
mRNA
5’
CUA
GAU
3’
tRNA
3’
5’
Wobble base pairing: base pairing between
the 3’ position of the codon and 5’ position
of the anticodon may occur by a nonstandard way. This allows one tRNA to
recognize more than one codon.
Examples of wobble base pairing
Anticodon wobble position base
C A G U
Codon wobble position base
G
I
U C A C
U G U
A
Wobble base pairing of inosine
with three nucleosides
rib o se
rib o se
rib o se
N
N
O
N
H
N
O
N
N
O
O
N
N
c ytid ine
Ino sine
O
N
N
N
rib o se
rib o se
N
N
N
H
H
N
N
N
H
H
H
Ino sine
N
H
H
N
N
N
O
N
rib o se
urid ine
Ino sine
a d e no sine
3) rRNAs and ribosomes: As the site of
protein biosynthesis, ribosome is made up
of two subunits, one is large and another is
small.
Composition of ribosomes in eukaryotes and
prokaryotes
Subunit size
Eukaryotic ribosome (80S)
Prokaryotic ribosome (70S)
large subunit small subunit
large subunit small subunit
60S
40S
50S
30S
rRNAs
5S, 5.8S, 28S
18S
5S, 23S
16S
Proteins
49
33
35
21
A) Polysomes: several ribosomes bind to and
translate a single mRNA molecule
simultaneously
B) Free ribosomes: ribosomes occur free in
the cytosol, usually synthesizing proteins of
cytosol, nucleus, mitochondria or other
organelles
C) Membrane bound ribosomes: ribosomes
bind to the membrane of rough endoplasmic
reticulum, usually synthesizing secretory
proteins or membrane proteins.
Polysomes
4) Aminoacyl-tRNA synthetase: is also called
amino acid activating enzyme, which
catalyzes the following reactions.
Synthetase
tRNA + Met
CH3
CH3
S
S
CH2
CH2
CH2
transformylase
O
H2N CH C
O
Met
(Met -tRNAi
tRNAf
Met
O
HC
CH2
O
HN CH C
O
tRNAf
)
Met
10
N -formyl tetrahydrofolate
(fMet-tRNAi
tetrahydrofolate
)
Met
2. Steps of Protein Biosynthesis
The steps of protein biosynthesis include:
initiation, elongation, and termination or
release.
1) Initiation: Translation begins with the
assembly of an initiation complex
consisting of an mRNA, a ribosome, and the
initiator tRNA (fMet-tRNAiMet or MettRNAiMet ) . The process requires a number
of protein factors, known as initiation
factors.
Formation of the initiation complex in
eukaryotic translation
mRNA
Cap
Cap
AUG
AUG
Met
Met
eIF1,2,3,4, GTP
60S + eIF5
Met
Cap
Cap
UAC
AUG
AUG
UAC
40S
ATP
ADP+Pi
eIF1,2,3,4,5, GDP+Pi
Cap
AUG
UAC
Met-tRNA
Initiation complex
In prokaryotes, initiation factors IF1 and
IF3 bind to the 30S subunit while IF2 binds
to GTP·fMet-tRNAiMet. The two complexes
and mRNA combine to form a pre-initiation
complex, releasing IF3. The 50S subunit
binds with this complex, with hydrolyzation
of the bound GTP to GDP and Pi, and
release of IF1 and IF2, to form a completed
initiation complex.
Formation of the initiation complex in
prokaryotic translation
IF2•GTP·fMet-tRNAiMet
30S
IF1, IF3
IF1 30S IF3
IF1 30S mRNA
mRNA
IF3
IF2•GTP·fMet-tRNAiMet
50S
mRNA
IF1, IF2, GDP
fMet
Prokaryotic and eukaryotic initiation factors
Prokaryotic eukaryotic
IF1
eIF 1
IF2
eIF2a eIF 2b eIF 2c
IF3
eIF3
eIF4a eIF4b eIF4c
eIF4d eIF4e eIF4f
eIF5
eIF6
Function
IF1 binds to small subunit before mRNA bi
nding. eIF1 assists
mRNA binding.
Bind initiator tRNA, stabilize ternary complex, cause GTP/GDP
exchange.
Bind to the small subunit, assist mRNA binding, cause
dissociation of subunits after translation.
Recognize and bind the mRNA cap, assist mRNA binding,
hydrolyze ATP to drive scanning for the initiator codon.
Promotes GTP hydrolysis and release of other initiator factors.
Assists subunit dissociation.
2) Elongation: Elongation of polypeptide
chain consists of a series of cycles, called
ribosomal cycles, each of which forms a
new peptide bond.
Three steps: entry, peptide bond formation,
and translocation.
A) Entry of aminoacyl-tRNA to the A site of ribosome
(A. in prokaryotes, B. in eukaryotes. AA = aminoacyl)
A.
Ts
T u -Ts + G T P
A A -tR N A
T u -G T P
GDP
A A -tR N A -T u -G T P
mRNA
Ts
Pi
T u -G D P
+
A A -tR N A -m R N A
B.
e E F -1 + G T P
e E F -1  
A A -tR N A
e E F -1  -G T P
GDP
A A -tR N A -e E F -1  -G T P
mRNA
e E F -1  
Pi
e E F -1  -G D P
+
A A -tR N A -m R N A
B) Peptide bond formation
dipeptidyl-tRNA
P site
A site
P site
N H2
CH
R1
H 2N
R2
CH
H 2N
CO
O
tRN A
1
R1
CO
Peptidyltransferase
NH
CH
CH
CO
OH
CO
O
tRN A 1
O
tRN A 2
A site
R2
tRN A 2
C) Translocation: Translocation is a process
involves the shift of the newly formed
peptidyl(n+1)-tRNA from the A site to the P
site, with release of the deacylated tRNA
from the ribosome. This process is mediated
by another elongation factor, EF-G in
prokaryotes, or eEF2 in eukaryotes.
The translocation step in protein biosynthesis
Peptidyl-tRNA
Peptidyl-tRNA
tRNA
P
5’
Translocation
A
P
5’
3’
GTP-EFG
EFG + GDP + Pi
A
3’
D) Termination: when a ribosome moves onto
the stop codon of mRNA, the stop codon in
the A site cannot be recognized by any
aminoacyl-tRNA molecules. Instead, release
factors interact with the mRNA-ribosome
complex, leading to discharge of the newly
synthesized polypeptide from the complex.
Termination of protein biosynthesis
NH 3+
Peptidyl-tRNA
NH3+
Peptidyl-tRNA
eRF-GTP
A
P
UAG
5’
UAG
5’
A
eRF-GTP
P
3’
3’
Pi
+
H 3N
Peptide chain
5’
40S
60S
tRNA
eRF-GDP
3’
tRNA
P
eIF6
eRF-GDP
5’
mRNA
UAG
A
3’
Elongation and termination factors in
prokaryotes and eukaryotes
Prokaryotic eukaryotic
Function
Elongation factors:
EFTu
eEF1
EFTs
eEF1
EFG
eEF2
Termination factors:
RF1
eRF
RF2
RF3
Bind amino acyl tRNA.GTP
Assist in the exchange of GTP and GDP
Hydrolyze GTP, translocate mRNA along ribosome
RF1 recognizes UAA, UAG. eRF recognizes all three
termination codons, and binds and hydrolyzes GTP,
causing release of peptide and tRNA from the ribosome.
Recognizes UAA, UGA.
Binds GTP and interacts with RF1 and RF2.
(Eukaryotic)
3. Posttranslational Processing
Newly synthesized polypeptides usually
undergo structural changes called
posttranslational processing.
The most important posttranslational
processing: modification and folding.
1) Posttranslational modification:
A) Modification of protein primary structures
 Removal of the N-terminal Met residue
H2 O
O
O
O
HC NH CH C NH CH C
CH2
¯
HCOO
O
AA3
AAn
+
COO
NH3
Deformylase
R
CH
CH2
CH2
CH2
SCH3
SCH3
Met-specific aminopeptidase
O
+
NH3 CH C
R
H2 O
Methionine
AA3
AAn
COO
C
O
NH
CH
R
C
AA3
AAn
COO
Posttranslational processing of human
preproinsulin
+
N H 3+
COO
H2O
H 3N
Signal peptide
S
S
S
S
COO
-
-
Signal peptidase
Proinsulin
Preproinsulin
6H2O
4AA + Peptide C
B chain
A chain
+
+
H 3N
H 3N
S
COO
S
S
S
Insulin
COO
-
-
B) Glycosylation: occurs in most membrane and
secretary proteins, such as glycoproteins.
Two types of glycoproteins in humans: O-linked
and N-linked. Formed in endoplasmic reticulum
and Golgi apparatus.
N-linked
O-linked
CH 2 OH
CH 2 OH
O

NH
NH
OH
CO
CH 2
CH
HO
O
NH
OH

CO
H O
NH
NH
CO
CO
CH 3
CH 3
O
CH 2
CH
CO
C) Modification of protein on higher-level
structures
Acetylation of the amino terminus:
Acetyl-SCoA + H2N-protein
Acetyl-NH-protein + HSCoA
Phosphorylation:
ATP
ADP
Protein kinase
Protein
Pi
Phosphoprotein
Phosphatase
H2O
2) Folding of newly synthesized polypeptides
Newly synthesized polypeptide chains usually
undergo folding, a process that requires
protein factors called molecular chaperones.
Two types of molecular chaperones:
chaperones and chaperonins.
The major function of molecular chaperones
is to assist the correct folding of nascent
polypeptide chains by blocking their
hopeless entangling or insignificant
intermolecular interactions.
Molecular chaperones belong to the “heatshock protein (HSP) family”.
A) Chaperone proteins: include HSP70,
HSP40, and GrpE.
 The binding-release cycle of chaperone
proteins with a nascent polypeptide earns
time for the proper folding of the unfolded
polypeptide chain. The cycle continues
until the polypeptide chain is folded to a
native conformation.
The binding-release cycle of a chaperon-polypeptide complex
Pi
ATP
HSP40-Polypeptide-HSP70-ADP
GrpE
ADP
HSP70-ATP
HSP40-Polypeptide
HSP40-Polypeptide-HSP70-ATP
HSP40
Polypeptide
Polypeptide
+ HSP70-ATP
B) Chaperonins: are also heat-shock proteins.
They participate in the folding of a variety
of proteins by forming a cylindrical
structure (a ring) enclosing a central cavity.
The target polypeptide chain enters the
central cavity of the folding machine, where
it is properly folded and is then released.
The entering-folding process repeats until a
native 3D structure of the protein is formed.
A folding cycle of a polypeptide by GroEL-GroES
chaperonins in E. coli cell
GroES
Folded polypeptide
polypeptide
+
GroEL
GroEL-polypeptide
PolypeptideGroEL-GroES complex
GroEL
GroES
4. Protein targeting
Protein targeting is a process in which a newly
synthesized protein is delivered to a specific
extracellular or intracellular location.
Secretory proteins are first synthesized by
ribosomes bound to the rough ER (RER),
with a signal sequence (or called signal
peptide) at the N-terminal end, which
directs the protein to be delivered to its
functioning place.
A.
Hydrophobic area
Signal peptidase
cleavage site
NSecretory protein
Signal peptide
Internal signal peptide
B.
N-
Signal peptide
Stop-transfer
sequence
Type III integral
membrane protein
(A) Signal peptides of secretory proteins. (B) Type III integral
membrane proteins with signal-peptide, internal signal-peptide,
and stop-transfer sequences.
The signal peptide directs a newly synthesized
secretary protein to enter into the RER lumen
SRP
RER lumen
SRP receptor
signal peptidase
Signal
peptide
RER membrane
SRP: signal recognition particle
5. Clinical correlation of protein biosynthesis
Protein biosynthesis is the means to express
the genes that control metabolisms in cells.
Any mistake occurs in protein biosynthesis
may result in severe consequences in
metabolism.
1) Molecular Diseases: refer to those resulting from
abnormal protein structures due to mutation of
genes.
 Sickle-cell anemia: a result from the replacement
of an amino acid residue at position 6 of the chain, glutamate , by another one, valine.
Position of -chain
Hemoglobin A
Hemoglobin S
1 2 3 4 5 6
7 8
Val-His-Leu-Thr-Pro-Glu-Glu-LysVal-His-Leu-Thr-Pro-Val-Glu-Lys-
2) Action of some antibiotics: they carry out
antimicrobial activities via inhibition of
protein synthesis in the microorganism,
such as tetracyclines, streptomycin,
chloramphenicol, and so on.
Some antibiotic inhibitors of protein biosynthesis
Antibiotic
Tetracycline
Streptomycin
Chloramphenicol
Erythromycin
(prokaryotes)
Puromycin
Cycloheximide
Action
Steps affected
Binds to the 30S subunit, inhibits
binding of aminoacyl-tRNA
Binds to the 30S subunit, inhibits
binding of fMet-tRNA and causes
misreading of mRNA
Binds to the 50S subunit, inhibits
peptidyltransferase activity
Binds to the 50S subunit
entry of aminoacyl-tRNA
(prokaryotes)
initiation, elongation
(prokaryotes)
Acts as an analog of aminoacyltRNA and binds to the A site
Blocks peptidyltransferase activity
Elongation (prokaryotes &
eukaryotes)
Elongation (eukaryotes)
Elongation (prokaryotes)
Translocation
3) Effect of some biological molecules :
Interferons (IFNs) are cytokines produced
during immune response to antigens,
especially to viral infections.
Two functions of IFNs: cause viral RNA
degradation and inhibit protein biosynthesis
in cells.
IFN
protein kinase
phosphorylation
of eIF-2a
inhibition of protein biosynthesis
in cells
inhibition of the viral replication.